DNA damage is a constant and considerable threat to genomic stability and cell survival. In response to genotoxic stresses that generate DNA double-strand breaks (DSBs), an evolutionary conserved protein complex known as Mre11-Rad50-Nbs1 (MRN) is critical for cell cycle checkpoint activation and DNA repair. Defects in these processes often lead to the development of cancer and diseases in humans. The Nbs1 subunit clearly regulates responses of the MRN complex to DNA damage, however the exact mechanisms of how it does so are not well understood. The amino-terminus of Nbs1 contains two phosphopeptidebinding motifs known as Forkhead-associated (FHA) and BRCA1 C-terminal (BRCT) domains. These domains are both mediators of phosphorylation-dependent protein-protein interactions, and are commonly found in proteins that regulate the cell cycle or DNA damage response. While some functional importance of these domains has been demonstrated for Nbs1, the specific phosphoprotein(s) that interact with them have not been clearly identified. To address this specific question the genetically tractable model organism Schizosaccharomyces pombe and a groundbreaking crystal structure of Nbs1 will be exploited to help identify novel interacting proteins of the Nbs1 FHA and/or BRCT domains. This problem will be attacked through multiple approaches including a high-copy suppressor genetic screen, yeast two-hybrid assay, and Multidimensional Protein Identification Technology (MudPIT) mass spectrometry. Newly identified Nbs1- interactors will be confirmed in vivo by co-immunoprecipitation and the functional implications of Nbs1 mutants defective in FHA- or BRCT-mediated protein-protein interactions will also be characterized, particularly their effects on: cellular DNA damage sensitivity, MRN complex stability, MRN complex association with DSBs, MRN complex mediation of ATM (Tell) activation, and telomeric stability. The research described in this proposal has a strong relevance to public health, as mutations leading to dysfunction of the Nbs1 protein are a direct cause of Nijmegen breakage syndrome in humans. Additionally, individuals afflicted with this condition are heavily predisposed to develop cancer as well. Therefore, identification of novel factors and processes that regulate the function of Nbs1 and the MRN complex has the potential to neutralize multiple human diseases.